There is an urgent need for better serological tests for the early detection, diagnosis and monitoring of hepatocellular carcinoma (HCC). The serological test that is currently used (alphafetoprotein, AFP) has limited sensitivity and specificity. Several studies have shown that a protein called Glypican-3 (GPC3) is a histological and serological marker of HCC. Using a genomics approach, we found GPC3 to be significantly over-expressed in HCC compared to non-tumor liver. In fact, GPC3 is second only to AFP in its magnitude of differential expression between HCC and non-tumor liver. Thus, we hypothesize that the combination of AFP and GPC3 measurements in serum could improve the ability to accurately detect HCC at its early stage, and therefore improve the efficacy of therapeutic interventions. Indeed, our preliminary results suggest that the simultaneous assessment of AFP and GPC3 could provide an improved test for HCC diagnosis and for the screening of patients at risk of developing this malignancy. The primary objective of this proposal is to improve and validate an ELISA for the detection of GPC3, and to use this assay to verify the clinical utility of combined analysis of GPC3 and AFP for the detection of HCC. While the methodology used to detect GPC3 is commonplace, the innovation lies in the discovery of it's specificity for HCC, and in the opportunity for combined analysis with another accepted biomarker (AFP) to improve the clinical utility of the test. We will accomplish the goals in Specific Aims I and 2 by generating monoclonal antibodies that pair well in ELISAs with the current antibody derived from the 1G12 hybridoma. In addition, refinements to the assay format will be made to improve the sensitivity of the GPC3 ELISA. In preliminary studies, we have already achieved at least a 10-fold increase in detection over the previous version of the test. We anticipate that we will develop an ELISA with picogram-level detection for GPC3 in serum, and that by increasing the sensitive range of the assay, greater sensitivity for detecting HCC can be achieved without sacrificing specificity. An additional goal of our proposal is to use this improved diagnostic test as a tool to monitor response to therapy. Since infection with Hepatitis B or Hepatitis C virus is a major risk factor for HCC, an improved diagnostic test for the early detection of this cancer could be a useful component of the management of this significant public health issue. [unreadable] [unreadable] [unreadable]